Organic acids have a long history of use as food additives, chelators, and dispersants and may also serve as platform chemicals, from which other useful chemicals are derived via simple chemical reactions. Currently many platform chemicals are derived from the petroleum industry, but as oil stocks become depleted it will be increasingly important to replace petroleum-derived chemicals with chemicals derived from alternative sources, such as plant biomass. Plant biomass is ligno-cellulosic and may contain a substantial fraction of xylose when it is hydrolysed.
The Sequence Listing submitted in text format (.txt) on Sep. 14, 2011, named “2081474US_ST25.txt”, (created on Tuesday, Sep. 13, 2011, 30.3 KB), is incorporated herein by reference.
Xylonic acid, as an organic acid, can be used in similar applications as gluconic acid but differs somewhat in its specific properties WO03/78347 (Chun et al.). Several applications for xylonic acid have been described including use as a precursor for 1,2,4-butanetriol synthesis (Niu et al. 2003 J Am Chem Soc. 2003 Oct. 29; 125(43):12998-9. Microbial synthesis of the energetic material precursor 1,2,4-butanetriol. Niu W, Molefe M N, Frost J W.), use to improve absorption of Vitamin C, WO90/03167 (Richard), use as a clarifier for polyolefins, U.S. Pat. No. 5,302,643 (Millner et al.), dispersal of concrete, WO03/78347 (Chun et al.), reduction of acrylamide in food cooked under heat, US2004/131737 (Tomoda et al.), and use as a biopesticide, WO2006/032530 (Pujos). However, the development of applications for xylonic acid has been limited by the lack of commercial supply and limited methods for producing xylonic acid.
Xylonate can be produced in high yields from D-xylose by Pseudomonas and Gluconobacter species, especially Pseudomonas fragi and Gluconobacter oxydans. D-Xylose is converted to xylono-γ-lactone by periplasmic xylose dehydrogenase and the xylono-γ-lactone is subsequently hydrolyzed either spontaneously or enzymatically to yield xylonate. Pre-treated ligno-cellulosic waste biomass can be used to produce xylonate using G. oxydans, and the use of Gluconobacter and other bacterial species for the conversion of sugars in ligno-cellulosic hydrolysates to aldonic acids, including the conversion of xylose to xylonic acid, has been described in WO03/78347 (Chun et al.). The use of G. oxydans and G. cerinus in the conversion of aldoses other than glucose to aldonic acid has been described in JP2007/028917 (Oe et al.).
WO2005/068642 and WO2008/091288 describe methods of producing 1,2,4 butanetriol enantiomers from xylose or xylonic acid containing starting material inside a transformed host cell. If the starting material is xylose, it is first converted to xylonic acid by xylose dehydrogenase and then further converted into 1,2,4 butanetriol by three other enzymes. The produced xylonate is not excreted but channeled inside the cell to subsequent reaction in the 1,2,4 butanetriol pathway.
Xylose dehydrogenase is naturally expressed in at least some bacteria. The xylose dehydrogenases found in Pseudomonas and Gluconobacter species are actually glucose dehydrogenases, which convert many other sugars than xylose and glucose to corresponding acids. Thus if the medium contains many different sugars, like ligno-cellulosic hydrolysates do, Gluconobacter produces many different acids, which can be difficult to separate. This is of course a drawback when only one specific acid is desired. In addition, high concentrations of ligno-cellulosic hydrolysate inhibit the growth and xylonic acid production of bacterial strains, and dilution or pretreatment of the hydrolysate is needed. Further the bacteria are relatively acid sensitive i.e. they do not grow at low pH.
The production of xylonate from xylose using a glucose oxidase or filamentous fungi producing glucose oxidase has been described in WO03/078347 (Chun et al.). No fungal system for bioproduction of xylonic acid with xylose dehydrogenase has been described so far.
Xylose dehydrogenase activity has been found in some eukaryotic organisms including a number of fungi such as Trichoderma viridea (Kanauchi M & Bamforth C W, 2003 J Inst Brew 109:203-207) and Pichia querquum (Suzuki T & Onishi H, 1973. Appl Microbiol 25:850-852). A xylose dehydrogenase encoding gene has recently been cloned from Trichoderma reesei (sexual/perfect state Hypocrea jecorina), and deposited in GenBank as EF136590. The gene was cloned into yeast, which was grown on a synthetic medium containing D-glucose as carbon source (Berghäll et al. 2007. FEMS Microbiol Lett 277: 249-253). After growth the yeast cells were disrupted and enzyme activity was measured from extracts of the disrupted cells by monitoring the formation of NADPH from NADP+ in the presence of D-xylose. The role of this enzyme was said to be unclear, because in T. reesei D-xylose is predominantly catabolised through a path with xylitol and D-xylulose as intermediates and the mould is unable to grow on D-xylonic acid. In some prokaryotic organisms D-xylose is catabolised using D-xylose dehydrogenase with D-xylonate as an intermediate. The D-xylose dehydrogenase of T. reesei was not believed to be part of such a pathway. Instead it was suggested that said dehydrogenase may play a role in the regeneration of the cofactor NADP+ in the presence of D-xylose.
The xylose dehydrogenase from T. reesei uses NADP+ as a co-factor. Other xylose dehydrogenases have been described which use both NADP+ and NAD+ as co-factors or primarily NAD+.
Many fungi are able to convert xylose to xylitol. Conversion of xylose to xylitol, with or without subsequent metabolism of the xylitol, represents a competing reaction for the conversion of xylose to xylonic acid. It has previously been shown that xylose reductase activity can be reduced or eliminated by the deletion of xylose and/or aldose reductases such as GRE3 in S. cerevisiae or xyrA in A. niger (Träff K L, et al., 2001. Appl Environ Microbial. December; 67(12):5668-74; Hasper A A et al., 2000. Mol Microbiol. April; 36(1):193-200).
There is presently a need for an improved method for producing xylonic acid without the drawbacks of the previously described bacterial processes. The present invention meets this need. Fungal species are in many cases the preferred industrial production organisms because of their low nutrition requirements, easy handling (rigid cell wall, big cells), inhibitor tolerance (e.g. present in lignocellulose hydrolysates), potential GRAS (generally regarded as safe) status and long history of usage e.g. in baking, brewing and acid production industries.
The present invention now provides a method for the manufacture of xylonic acid, which is especially suited for the biorefinery concept i.e. utilization of biomass. The method may be carried out at low pH. A further advantage is that a high concentration of ligno-cellulosic hydrolysate can be used, which makes the method economic. The method is especially convenient in that the xylonic acid can be recovered directly from the spent culture medium, without the need to disrupt the cells first. Further the method is highly specific in that production of other non-desired acids is negligible.